Zebrafish Neuronal Nicotinic Acetylcholine Receptors: Cloning, Expression, and Functional Analysis DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Kristin Michelle Ackerman ***** The Ohio State University 2009 Dissertation Committee: Approved by Professor Robert Thomas Boyd, Advisor Professor Christine Beattie _________________________________ Professor Dennis McKay Advisor Professor James Jontes Integrated Biomedical Sciences ABSTRACT Cigarette smoking is a major public health concern, in that, over one billion people or one in three adults in the world smoke (WHO, 2006). Even more alarming is that approximately 20% of pregnant women in the western world continue to smoke during pregnancy despite a number of fetal complications linked with cigarette smoking (CDC, 2004, 2006 and U.S. Surgeon General’s Report (Chapter 5; pages 180–194). Smoking during pregnancy can cause serious health problems for both mother and child, such as pregnancy complications, premature birth, low-birth-weight infants, stillbirth and infant death (U.S. Department of Health and Human Services, 2004). Nicotine exposure in utero can also result in long-term anatomical, behavioral and cognitive deficits (Sexton et al., 1990; Olds et al., 1994; Slotkin 1992, 2004, 2008). Nicotine, the main addictive chemical component in cigarette tobacco, is a likely contributor to the adverse outcomes, as fetal brain and spinal cord are primary targets. Nicotine mediates its actions through nicotinic acetylcholine receptors, nAChRs, which are widely distributed throughout the body. This class of receptors belongs to the ligand gated ion channel superfamily, which includes GABA A, glycine, 5-HT3, and NMDA-type glutamate receptors. The nAChR is widely distributed in a vast majority of organisms and can be classified into two groups, muscle or neuronal. The muscle type is found at the neuromuscular junction (NMJ), while the neuronal type is found in nervous tissue. Each nAChR, despite its localization, is composed of five subunits that assemble around a central channel. To date many disease states have been associated with the nAChRs in adult including Alzheimer’s disease, Parkinson’s disease, schizophrenia, Tourette’s syndrome, ii epilepsy, and nicotine addiction. By conducting studies in a developing animal we are addressing the one million pregnant women in the United States and 20% of pregnant women in the western world that continue to smoke regardless of the harm they may be causing the fetus. The primary function of nAChRs is to modulate synaptic transmission. However, findings over the last 20 years indicate that neuronal nAChRs may play a potential role in the development of the nervous system. To date, it remains unclear as to how specific nAChRs subtypes function during normal developmental processes and how exposing the fetus to chronic nicotine affects fetal development. The zebrafish (Danio rerio) has shown incredible potential for the examination of events that occur during development. The results from our laboratory presented in this manuscript represent a continuation in the study of neuronal nAChR in the vertebrate zebrafish model. Our research efforts have resulted in the cloning of zebrafish chrna4 and chrna6 neuronal nAChR genes. Full-length cDNAs for these zebrafish neuronal nAChR subunit genes were determined and sequence analysis showed that these cDNAs demonstrated a high degree of homology to nAChR genes found in other species. To date, it does not appear that the chrna4 and chrna6 genes were duplicated in the zebrafish genome. Additionally, our laboratory has determined RNA expression patterns and examined the functional roles that nAChRs may play in nervous system development. Time course studies using RT-PCR and subunit-specific primers to each of the α2, α4, and α6 subunits determined when each gene was expressed. All of these subunits were expressed very early in development, with chrna2 RNA being expressed maternally in unfertilized embryos, chrna4 RNA expressed by 3 hours post fertilization (hpf), and chrna6 RNA present by 10 hpf. iii In situ hybridization studies using sense and antisense digoxigenin-labeled RNA probes were also used to localize the temporal and spatial expression of nAChR RNA in developing embryos. Each of the subunits displayed unique expression patterns that were transiently regulated. The zebrafish chrna4 RNA was expressed in a subset of hindbrain neurons in rhombomeres 4-7 and in cells consistent with dlx2 expressing neural crest cells migrating along the pharyngeal arch at 24 hpf. Additionally, limited expression was observed in forebrain and midbrain structures. At 48 hpf significant bilateral expression was seen in both midbrain and hindbrain consistent with the nucleus of the medial longitudinal fascicle and reticulospinal neurons, with no expression detected in the spinal cord. At 72 and 96 hpf, chrna4 continued to be highly expressed in specific midbrain and hindbrain areas. At 24 hpf, zebrafish chrna6 RNA was expressed in a subset of Rohon Beard sensory neurons, ventral neurons in the spinal cord, diencephalon, trigeminal ganglion, pineal, and in the first hindbrain rhombomere ventral to the cerebellum. At 48 hpf chrna6 was no longer observed in spinal neurons, but was still expressed in trigeminal ganglion, pineal, and was now co-localized to the TH + locus coeruleus and the midbrain diencephalic catecholaminergic cluster. chrna6 was highly expressed in retina at 48 hpf and minimally expressed in tectum which was not detected 24 hpf. At 72 and 96 hpf, zebrafish chrna6 continued to be expressed in trigeminal ganglion, retina, and pineal. chrna6 RNA was highly expressed in tectum in 72 and 96 hpf zebrafish, at robust levels with more widespread distribution than at 48 hpf. Additionally, at 96 hpf chrna6 expression was detected for the first time with a pattern consistent with cranial sensory neurons in the hindbrain. At 72 hpf and 96 hpf chrna6 RNA continued to be expressed in iv the diencephalic catecholaminergic cluster, but was also present in non- catecholaminergic cells in both midbrain and hindbrain. At 10 hpf, around the time of somitogenesis, chrna2 expression was heavy in the anterior head region with some labeling in the dorsal spine. By 18 hpf, there was diffuse labeling in the brain and a well defined pattern in the anterior spinal cord. At 24 hpf chrna2 was expressed in the forebrain consistent with the olfactory bulb, interneurons in the first 1-7 hemisegments of the spinal cord, interneurons localized down the entire length of the spinal cord, and cells along the midbrain/hindbrain boundary. At 48 hpf chrna2 forebrain expression had disappeared and midbrain expression was prominent with very limited expression in hindbrain. The distinct pattern in the spinal cord remained intact. By 72 hpf all the spinal cord labeling had disappeared, but robust expression remained in the midbrain with limited expression in the hindbrain. By 96 hpf chrna2 expression was still evident in the midbrain. An antisense oligonucleotide morpholino gene knock down approach was used to determine the function of chrna2 during development. Knock down of the chrna2 resulted in swimming deficits, paralysis, and a disruption in the number, morphology, and extenstion of dorsal projecting motor neurons to the dorsal myotome. The loss of chrna2 did not affect ventral projecting motor neurons. Expression of the zebrafish nAChR α2 subunit RNA in Xenopus oocytes provided preliminary evidence of functional nAChRs in the zebrafish model. Whole-cell recordings demonstrated that the zebrafish nAChR α2 subunit was able to assemble as a heteromeric receptor and, upon activation, produced currents characteristic of α2 nAChRs in other species. v Treatment of zebrafish with nAChR agonists and antagonists was done to further examine the effects activating or blocking nAChRs on development. Treatment of zebrafish embryos with 50-200 µM concentrations of the nAChR agonist nicotine during specific periods of development resulted in motor behavior deficits, paralysis, and the induction of apoptosis. This nicotine-induced cell death was blocked when the nAChR antagonist, DHβE, was co-applied with nicotine, confirming this mechanism involved nAChRs. Embryos treated with nicotine displayed a nicotine-mediated upregulation of chrna2, chrna 4, chrna 6, chrna7, cfos, p53, islet 1 and shh transcripts. In addition to the in vivo work using zebrafish embryos, preliminary screening the zebrafish cell line, ZEM2S, indicated that α2, α4, α6, α7, and β2 nAChR subunit RNAs, along with shh, islet 1, p53 and cfos were present. α3 and α8 RNAs were not detected. β2 and β4 nAChR RNAs weret been screened. The expression of these genes in cell culture perfectly modeled the nAChR expression in 10 hpf zebrafish embryos where α2, α4, α6, α7, β2, and β3 nAChRs, along with Shh, Islet 1, p53 and cfos were also expressed. Preliminary studies indicate that nicotine induced upregulation of cfos transcript is evident in the cell line after 12 or 24 hours of drug exposure. Although our preliminary work has generated a substantial amount of data in several areas, much work remains to be done in the zebrafish model. Future plans using the zebrafish to study nAChRs include the determination of expression patterns of the remaining nAChR subunits, the use of morpholino oligonucleotides to detect the functions of specific nAChRs subtypes, and the examination of the effect of other cholinergic agents on nervous system development. Our results indicate that zebrafish vi will provide an excellent model for the continued study of the role of cholinergic receptors in neural development. vii DEDICATION I would like to dedicate this work to my family for all of their love and support. viii ACKNOWLEDGMENTS Foremost, I wish to acknowledge my advisor, Dr. R. Thomas Boyd for all of his patience, guidance, and support. He has provided me with invaluable neuromolecular biology training and I am forever grateful. I would also like to acknowledge the support of my colleagues Erin Fink, Jenn Stanke, Jona Hilario, and all the members of the Beattie and Henion labs.